Immunopotentiator-linked oligomeric influenza immunogenic compositions

ABSTRACT

Disclosed herein are immunogenic compositions for preventing infection with influenza viruses wherein the immunogenic compositions comprises an immunogen such as a hemagglutinin of an influenza virus, and an immunopotentiator such as an Fc fragment of human IgG and optionally a stabilization sequence. The immunogen is linked to the stabilization sequence which in turn is linked to the immunopotentiator.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No. 12/897,997, filed Oct. 5, 2010, which claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 61/250,442 filed Oct. 9, 2009, the entire contents of all of which are incorporated by reference herein.

FIELD OF THE INVENTION

The present disclosure relates to the field of immunogenic compositions for the prevention of influenza infection.

BACKGROUND OF THE INVENTION

The Influenza A virus, which belongs to the Orthomyxoviridae family, can cause influenza in humans, birds or domesticated food animals. The virus can be classified into different subtypes based on their surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Of the 16 known HAs and nine NAs, three HA subtypes (H1, H2, and H3) and two NA subtypes (N1 and N2) are most commonly found in humans. H1N1 and H3N2 are the major subtypes that cause human seasonal flu and global pandemics of influenza. The influenza pandemic in 2009 is caused by influenza A virus H1N1 of swine origin. This has led to a growing concern regarding the pandemic potential of the highly pathogenic avian influenza (HPAI) H5N1 viruses. Thus the development of an effective and safe vaccine against divergent influenza A virus strains is urgently needed for the prevention of future outbreaks of influenza.

SUMMARY OF THE INVENTION

Disclosed herein are immunogenic compositions for the prevention of infection with influenza viruses. The disclosed immunogenic compositions are trimeric proteins comprising: 1) an immunogen, such as an influenza hemagluttinin sequence; 2) a trimerization or stabilization sequence; and 3) an immunopotentiator sequence. The three sequences are contiguous and expressed as a single protein in a mammalian expression system or the immunogen and the immunogen and the immunopotentiator are chemically linked and stabilized.

In one embodiment, disclosed herein is an immunogenic composition for induction of an immune response against influenza virus, the immunogenic composition comprising a polypeptide comprising an immunogen and an immunopotentiator. In another embodiment, the polypeptide further comprises a stabilization sequence.

In other embodiments, the immunogen is a hemagglutinin sequence of an influenza virus, a neuraminidase sequence of an influenza virus or a membrane protein sequence of an influenza virus. In another embodiment, the immunogen is a fragment of said hemagglutinin sequence selected from the group consisting of HA1, HA2 and RBD. In another embodiment, the immunopotentiator is selected from the group consisting of the Fc fragment of human IgG, C3d, Onchocerca volvulus ASP-1, cholera toxin and muramyl peptides.

In another embodiment, the stabilization sequence is foldon or GCN4.

In yet another embodiment, the polypeptide is a fusion protein. In another embodiment, the polypeptide is produced in a mammalian expression system.

In another embodiment, the polypeptide is selected from the group consisting of HA1-hFc, HA-hFc, HA1+3-259-hFc, HA1-Fd-hFc, HA-Fd-hFc, HA2-Fd-hFc, HA-RBD-Fd-hFc, and HA1+3-259-Fd-hFc.

In another embodiment, the immunogen is linked to said stabilization sequence and wherein said stabilization sequence is linked to the immunopotentiator in a single polypeptide. In yet another embodiment, the immunogen and the immunopotentiator are chemically stabilized by 2,2-bipyridine-5-carboxylic acid (BPY).

In still another embodiment, the immunogenic composition further comprises an adjuvant.

Also disclosed herein is a method of inducing a protective immune response against an influenza virus, the method comprising administering the immunogenic composition of claim 1 to a host in need thereof and wherein the immunogenic composition induces a protective immune response against challenge with an infectious agent in the host. In another embodiment, the immunogenic composition further comprises an adjuvant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the structure of the hemagluttinin (HA) protein of an influenza A H5N1 virus [A/Anhui/1/2005(H5N1)] and the construction of the recombinant HA1-hFc and HA1-Fd-hFc proteins containing the HA1 fragment (amino acids [aa] +3-322), and the recombinant HA-3-259-Fd-hFc protein including HA1 fragment of aa+3-259 of H5N1 virus HA fused to human IgG Fc (hFc), with or without the Fd sequence. The protease cleavage site RERRRKR between HA1 and HA2 is set forth as SEQ ID NO:41.

FIG. 2 depicts the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (FIG. 2a ) and Western blot (FIG. 2b ) analyses of the expressed HA1-hFc and HA1-Fd-hFc proteins.

FIG. 3 depicts Fast Protein Liquid Chromatography (FPLC) analysis of the expressed HA1-hFc and HA1-Fd-hFc proteins. The molecular weight of the proteins was indicated on each peak, with the structure corresponding to calculated standard proteins.

FIG. 4 depicts the immunization scheme of BALB/c mice with recombinant HA1-hFc, HA1-Fd-hFc and HA-3-259-Fd-hFc proteins, detection of induced antibodies and neutralizing activity, and challenge of the vaccinated mice with live H5N1 virus for cross-protection evaluation.

FIG. 5 depicts the binding reactivity of mouse sera (1:3,000 dilution) collected at day 0 (pre-immune) and 10 days post the 1^(st), 2^(nd) and 3^(rd) boosts with recombinant HA1-hFc and HA1-Fd-hFc proteins.

FIG. 6 depicts the ability of IgG antibody (Ab) to bind to HA1-hFc, HA1-Fd-hFc and HA-3-259-Fd-hFc fusion proteins detected in sera of mice collected 10 days post last vaccination with HA1-hFc, HA1-Fd-hFc and HA-3-259-Fd-hFc proteins.

FIG. 7 depicts the mean anti-HA1-hFc, anti-HA1-Fd-hFc and anti-HA-3-259-Fd-hFc IgG Ab titer of mouse sera collected 10 days post last vaccination.

FIG. 8 depicts the ability of IgG Ab to bind to an HA1 protein without Fd and Fc, detected in serially diluted sera of mice collected 10 days post last vaccination with HA1-hFc, HA1-Fd-hFc and HA-3-259-Fd-hFc proteins.

FIG. 9 depicts the ability of IgG Ab binds to A/VietNam/1194/2004 (VN/1194)-inactivated H5N1 virus, detected in serially diluted sera of mice collected 10 days post last vaccination with HA1-hFc, and HA1-Fd-hFc.

FIG. 10 depicts ability of IgG1 subtype Ab to bind to the HA1 protein, detected in serially diluted sera of mice collected 10 days post last vaccination with HA1-hFc, and HA1-Fd-hFc.

FIG. 11 depicts ability of IgG2a subtype Ab to bind to the HA1 protein, detected in serially diluted sera of mice collected 10 days post last vaccination with HA1-hFc, and HA1-Fd-hFc.

FIG. 12 depicts the comparison of IgG1 and IgG2a Ab responses in HA1-hFc- and HA1-Fd-hFc-vaccinated mice.

FIG. 13 depicts the neutralizing Ab titers (NT₅₀)) of sera from mice after each boost vaccination with HA1-hFc against H5N1 pseudovirus expressing XJ-HA, QH-HA, AH-HA and HK-HA as measured by pseudovirus neutralization assay.

FIG. 14 depicts the neutralizing Ab titers (NT₅₀)) of sera from mice after each boost vaccination with HA1-Fd-hFc against H5N1 pseudovirus expressing XJ-HA, QH-HA, AH-HA and HK-HA as measured by pseudovirus neutralization assay.

FIG. 15 depicts the neutralizing Ab titers (NT₅₀)) of sera from mice after each boost vaccination with HA1-hFc and HA1-Fd-hFc against H5N1 pseudovirus expressing homologous AH-HA as measured by pseudovirus neutralization assay.

FIG. 16 depicts the neutralizing Ab titers (NT₅₀)) against HA of heterologous (HK-HA, 1194-HA, QH-HA and XJ-HA) and homologous (AH-HA) strains of H5N1 pseudovirus, detected in sera of mice at 10 days post last vaccination with HA1-hFc, and HA1-Fd-hFc.

FIG. 17 depicts the neutralizing Ab titers (NT₅₀)) against HA of heterologous (HK-HA, 1194-HA) and homologous (AH-HA) strains of H5N1 pseudovirus, detected in sera of mice at 10 days post last vaccination with HA-3-259-Fd-hFc protein.

FIG. 18 depicts the neutralizing Ab titers (NT₅₀)) against heterologous strains A/Hong Kong/156/97 (HK/156), VN/1194 and A/Shenzhen/406H/06 (SZ/406H) of H5N1 live virus, detected in sera of mice at 10 days post last vaccination with HA1-hFc, and HA1-Fd-hFc.

FIG. 19 depicts the hemagglutination inhibition (HI) antibody titers against heterologous strains (HK/156, VN/1194 and SZ/406H) of H5N1 live virus, detected in sera of mice at 10 days post last vaccination with HA1-hFc, and HA1-Fd-hFc.

FIG. 20 depicts the cross-protection of HA1-hFc- and HA1-Fd-hFc-vaccinated mice against lethal H5N1 virus challenge, indicated by survival rate (%) of mice challenged with HK/156 strain (clade 0) of H5N1 live virus,

FIG. 21 depicts the cross-protection of HA1-hFc- and HA1-Fd-hFc-vaccinated mice against lethal H5N1 virus challenge, indicated by survival rate (%) of mice challenged with VN/1194 strain (clade 1) of H5N1 live virus.

FIG. 22 depicts the cross-protection of HA1-hFc- and HA1-Fd-hFc-vaccinated mice against lethal H5N1 virus challenge, indicated by survival rate (%) of mice challenged with SZ/406H strain (clade 2.3.4) of H5N1 live virus.

FIG. 23 depicts the quantification of viral RNA in lung tissue of H5N1 virus-challenged mice by quantitative reverse transcription PCR (QRT-PCR). Viral titers of HK/156, VN/1194 and SZ/406H strains of H5N1 virus were determined in lung tissues of the mice vaccinated with HA1-hFc and HA1-Fd-hFc proteins.

FIG. 24 depicts the evaluation of histopathological changes in the lung tissues of HA1-hFc- and HA1-Fd-hFc-vaccinated mice following lethal challenge with heterologous strains of H5N1 virus. Lung tissues from mice injected with PBS and those of uninfected mice were used as negative and normal controls, respectively. All sections of lung tissues were stained with hematoxylin and eosin (H&E) and observed under a light microscope (magnification, 100×). Representative images of histopathological damage from vaccinated mice challenged with H5N1 strains HK/156 (a), VN/1194 (b), and SZ/406H (c) are depicted.

DEFINITION OF TERMS

To facilitate an understanding of the following Detailed Description, Examples and appended claims it may be useful to refer to the following definitions. These definitions are non-limiting in nature and are supplied merely as a convenience to the reader.

Gene: A “gene” as used herein refers to at least a portion of a genetic construct having a promoter and/or other regulatory sequences required for, or that modify the expression of, the genetic construct.

Host: As used herein “host” refers to the recipient of the present immunogenic compositions. Exemplary hosts are mammals including, but not limited to, primates, rodents, cows, horses, dogs, cats, sheep, goats, pigs and elephants. In one embodiment of the present invention the host is a human. For the purposes of this disclosure host is synonymous with “vaccinee.”

Immunogen: As used herein the term “immunogen” refers to any substrate that elicits an immune response in a host. Immunogens of the present disclosure include, but are not limited to hemaglutinins of influenza viruses.

Immunogenic Composition: As used herein an “immunogenic composition” refers to an expressed protein or a recombinant vector, with or without an adjuvant, which expresses and/or secretes an immunogen in vivo and wherein the immunogen elicits an immune response in the host. The immunogenic compositions disclosed herein may or may not be immunoprotective or therapeutic. When the immunogenic compositions may prevent, ameliorate, palliate or eliminate disease from the host then the immunogenic composition may optionally be referred to as a vaccine. However, the term immunogenic composition is not intended to be limited to vaccines.

DETAILED DESCRIPTION OF THE INVENTION

Development of an effective and safe vaccine against divergent influenza A viruses is urgently needed for the prevention of future outbreak of influenza, especially the pandemic potential of the divergent strains of highly pathogenic avian influenza (HPAI) H5N1 viruses. The present disclosure describes the development of a subunit influenza vaccine based on the surface hemagglutinin (HA) proteins of an influenza A virus. This candidate vaccine uses mammalian cell-expressed recombinant proteins encoding the HA1 fragment of HA. It induced strong immune responses, potent neutralizing antibodies and extensive cross-protective immunity in vaccinated animals. The elicited neutralizing antibodies were proven to be effective against at least five strains of pseudotyped influenza A virus isolates representing clades 0, 1, 2.2, and 2.3, and neutralize and cross-protect against at least three strains of live H5N1 influenza viruses covering clade 0, 1 and 2.3.4.

In one embodiment disclosed herein, provided is a subunit influenza vaccine (immunogenic composition) comprising a hemagglutinin (HA) of an influenza virus, a stabilization sequence and an immunopotentiator. In another embodiment, the immunogenic composition is expressed in a mammalian expression system.

A universal influenza vaccine that could provide heterosubtypic immunity would be a tremendous advance for public health. Disclosed herein is a candidate influenza vaccine, using mammalian 293T cell-expressed fusion proteins encoding the HA1 fragment (residues +3-322, SEQ ID NO. 2, and residues +3-259, SEQ ID NO. 30, respectively) of an influenza A H5N1 virus [A/Anhui/1/2005(H5N1)]. The expressed recombinant protein was fused with a foldon (Fd) sequence (SEQ ID NO. 6) and the Fc fragment (SEQ ID NO. 7) of human IgG1 (hFc), with the purpose to maintain the trimerization structure of native HA proteins and to increase the stability and immunogenicity. Foldon is a trimerization or oligomerization motif from the T4 bacteriophage fibritin. HA is a homotrimeric integral membrane glycoprotein. It is shaped like a cylinder, and is approximately 13.5 nanometers long. The three identical monomers that constitute HA are constructed into a central a helix coil; three spherical heads contain the sialic acid binding sites. HA monomers are synthesized as precursors that are then glycosylated and cleaved into two smaller polypeptides: the HA1 and HA2 subunits. Each HA monomer consists of a long, helical chain anchored in the membrane by HA2 and topped by a large HA1 globule.

In one embodiment, the immunopotentiator is an immunoglobulin Fc fragment. The immunoglobulin molecule consists of two light (L) chains and two heavy (H) chains held together by disulfide bonds such that the chains form a Y shape. The base of the Y (carboxyl terminus of the heavy chain) plays a role in modulating immune cell activity. This region is called the Fc (fragment, crystallizable) region, and is composed of two heavy chains that contribute two or three constant domains depending on the class of the antibody. By binding to specific proteins, the Fc region ensures that each antibody generates an appropriate immune response for a given antigen. The Fc region also binds to various cell receptors, such as Fc receptors, and other immune molecules, such as complement proteins. By doing this, it mediates different physiological effects including opsonization, cell lysis, and degranulation of mast cells, basophils and eosinophils.

The disclosed immunogenic compositions have high efficacy in inducing potent immune responses in tested animals. They are able to elicit highly potent neutralizing antibodies that could neutralize not only homologous A/Anhui/1/2005 (AH, clade 2.3) strains but also heterologous A/Hong Kong/156/97 (HK, clade 0), A/VietNam/1194/2004 (1194, clade 1), A/Xinjiang/1/2006 (XJ, clade 2.2), and A/Qinghai/59/05 (QH, clade 2.2) strains of H5N1 viruses expressing HA proteins in a cell culture-based pseudovirus neutralizing assay. In addition, the disclosed immunogenic compositions highly neutralize and completely cross-protect against at least three divergent strains of H5N1 live viruses, including clade 0: A/Hong Kong/156/97 (HK/156), clade 1: A/VietNam/1194/2004 (VN/1194) and clade 2.3.4: A/Shenzhen/406H/06 (SZ/406H). The above features demonstrate that the expressed fusion proteins have a high potential to be developed into a universal influenza vaccine for the prevention of future flu outbreaks.

Previously designed influenza HA-based vaccines could not induce highly potent and broad neutralizing responses in the hosts, most likely because these vaccines could not properly maintain the stable and soluble trimeric conformation, or they lack efficient immunogenicity to induce high levels of neutralizing antibodies. The presently described immunogenic compositions have solved these problems by: 1) addition of Fd, a trimerization motif, to HA1 allows the HA1 to properly maintain the stable and soluble trimeric conformation; and 2) fusion of Fc fragment of IgG to HA1-Fd which results in enhanced immunogenicity of HA1-trimer to induce high levels of neutralizing antibodies and cross-protection against a broad spectrum of influenza viruses. In addition, the Fc fragment has tendency to form a non-covalent dimer through its disulfide bond, which may allow the fusion protein to form a dimmer, hexamer or other form of oligomer, resulting in a more immunogenic molecule.

The ability to induce antibodies against divergent strains of a particular virus would solve the one strain-one vaccine problem that has been a significant hurdle for all manufacturers of flu vaccines. Furthermore, the described formulation does not utilize chicken eggs to grow the virus—a major advantage that not only significantly reduces manufacturing time and cost but also allows pregnant women and persons allergic to chicken egg proteins to receive the vaccine.

TABLE 1 Components that can be used for design of immunopotentiator- linked oligomeric Influenza vaccines Stabilization molecule Proteins of influenza or method to form Immunopotentiators viruses* trimer or oligomer or modulators Hemagglutinin (HA) GCN4 cholera toxin HA1 Foldon immunomodulators (such as cytokines) HA2 2,2-bipyridine-5- bacterial LPS carboxylic acid (BPY) Peptides from HA1 Disulfide bonds Synthetic LPS mimetic RC529 Peptides from HA2 Facile ligation muramyl peptides Neuraminidase (NA) Monophosphoryl lipid A (MPL) Peptides from NA dsRNA complexes Membrane protein (M) CpG ODN, CTA1-DD Peptides from M IgG Fc HA receptor binding C3d domain (RBD) ASP-1 TGF-β or Th2 cytokines *including different subtypes.

In one embodiment, the influenza virus component of the instant immunogenic composition can comprise a sequence selected from the group consisting of the HA sequence of influenza virus H5N1; the HA sequence of influenza virus H1N1; the HA sequence of influenza virus H3N2; the HA1 sequence of influenza virus H5N1; the HA1 sequence of influenza virus H1N1; the HA1 sequence of influenza virus H3N2; the HA2 sequence of influenza virus H5N1; the HA2 sequence of influenza virus H1N1; the HA2 sequence of influenza virus H3N2; the NA sequence of influenza virus H5N1; the NA sequence of influenza virus H1N1; the NA sequence of influenza virus H3N2; the M1/M2 sequence of influenza virus H5N1; the M1/M2 sequence of influenza virus H1N1; the M1/M2 sequence of influenza virus H3N2; the HA-NA sequence of influenza virus H5N1; the HA-NA sequence of influenza virus H1N1; the HA-NA sequence of influenza virus H3N2; the HA-M1/M2 sequence of influenza virus H5N1; the HA-M1/M2 sequence of influenza virus H1N1; the HA-M1/M2 sequence of influenza virus H3N2; the HA-NA-M1/M2 sequence of influenza virus H5N1; the HA-NA-M1/M2 sequence of influenza virus H1N1; and the HA-NA-M1/M2 sequence of influenza virus H3N2. Amino acid and nucleic acid sequences for each of the above domains can be found in the Influenza Research Database.

TABLE 2 Amino acid and DNA sequences of immunopotentiator-linked oligomeric influenza immunogenic compositions (Note: SEQ ID NO. 40 and 42-48 are DNA sequences, while others are amino acid sequences)  SEQ ID NO. 1 [A/Anhui/1/2005(H5N1) HA]:  MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPL ILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKI QIIPKSSWSDHEASSGVSSACPYQGTPSFFRNVVWLIKKNNTYPTIKRSYNNTNQEDLLILWGIH HSNDAAEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQNGRMDFFWTILKPNDAINFES NGNFIAPEYAYKIVKKGDSAIVKSEVEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSN KLVLATGLRNSPLRERRRKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKEST QKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENE RTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEAR LKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICI SEQ ID NO. 2 [A/Anhui/1/2005(H5N1) HA1 +3-322]:  ICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPM CDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGV SSACPYQGTPSFFRNVVWLIKKNNTYPTIKRSYNNTNQEDLLILWGIHHSNDAAEQTKLYQNPT TYISVGTSTLNQRLVPKIATRSKVNGQNGRMDFFWTILKPNDAINFESNGNFIAPEYAYKIVKKG DSAIVKSEVEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNKLVLATGLRNSPL SEQ ID NO. 3 [A/Anhui/1/2005(H5N1) HA2]:  GLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQ FEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQ LRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSI YSTVASSLALAIMVAGLSLWMCSNGSLQCRICI SEQ ID NO. 4 [A/Anhui/1/2005(H5N1) HA-RBD]:  LSRINHFEKIQIIPKSSWSDHEASSGVSSACPYQGTPSFFRNVVWLIKKNNTYPTIKRSYNNTNQ EDLLILWGIHHSNDAAEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQNGRMDFFWTILK PNDAINFESNGNFIAPEYAYKIVKK SEQ ID NO. 5 [IL2ss signal peptide]:  MYRMQLLSCIALSLALVTNS  SEQ ID NO. 6 [Foldon (Fd), also see SEQ ID NO. 36]:  GYIPEAPRDGQAYVRKDGEWVLLSTFL SEQ ID NO. 7 IgG Fc (hFc)1:  RSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 8  MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPL ILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKI QIIPKSSWSDHEASSGVSSACPYQGTPSFFRNVVWLIKKNNTYPTIKRSYNNTNQEDLLILWGIH HSNDAAEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQNGRMDFFWTILKPNDAINFES NGNFIAPEYAYKIVKKGDSAIVKSEVEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSN KLVLATGLRNSPLRERRRKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKEST QKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENE RTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEAR LKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICIGYIPEAPRDGQ AYVRKDGEWVLLSTFL SEQ ID NO. 9  ICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPM CDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGV SSACPYQGTPSFFRNVVWLIKKNNTYPTIKRSYNNTNQEDLLILWGIHHSNDAAEQTKLYQNPT TYISVGTSTLNQRLVPKIATRSKVNGQNGRMDFFWTILKPNDAINFESNGNFIAPEYAYKIVKKG DSAIVKSEVEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNKLVLATGLRNSPL-GYI PEAPRDGQAYVRKDGEWVLLSTFL SEQ ID NO. 10  GLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQ FEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQ LRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSI YSTVASSLALAIMVAGLSLWMCSNGSLQCRICI-GYIPEAPRDGQAYVRKDGEWVLLSTFL SEQ ID NO. 11  LSRINHFEKIQIIPKSSWSDHEASSGVSSACPYQGTPSFFRNVVWLIKKNNTYPTIKRSYNNTNQ EDLLILWGIHHSNDAAEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQNGRMDFFWTILK PNDAINFESNGNFIAPEYAYKIVKK-GYIPEAPRDGQAYVRKDGEWVLLSTFL SEQ ID NO. 12 [HA-hFc]:  MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPL ILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKI QIIPKSSWSDHEASSGVSSACPYQGTPSFFRNVVWLIKKNNTYPTIKRSYNNTNQEDLLILWGIH HSNDAAEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQNGRMDFFWTILKPNDAINFES NGNFIAPEYAYKIVKKGDSAIVKSEVEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSN KLVLATGLRNSPLRERRRKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKEST QKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENE RTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEAR LKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICI-RSDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 13  ICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPM CDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGV SSACPYQGTPSFFRNVVWLIKKNNTYPTIKRSYNNTNQEDLLILWGIHHSNDAAEQTKLYQNPT TYISVGTSTLNQRLVPKIATRSKVNGQNGRMDFFWTILKPNDAINFESNGNFIAPEYAYKIVKKG DSAIVKSEVEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNKLVLATGLRNSPL-RS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 14  GLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQ FEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQ LRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSI YSTVASSLALAIMVAGLSLVVMCSNGSLQCRICI-RSDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK SEQ ID NO. 15  LSRINHFEKIQIIPKSSWSDHEASSGVSSACPYQGTPSFFRNVVWLIKKNNTYPTIKRSYNNTNQ EDLLILWGIHHSNDAAEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQNGRMDFFWTILK PNDAINFESNGNFIAPEYAYKIVKK-RSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK SEQ ID NO. 16  MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPL ILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKI QIIPKSSWSDHEASSGVSSACPYQGTPSFFRNVVWLIKKNNTYPTIKRSYNNTNQEDLLILWGIH HSNDAAEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQNGRMDFFWTILKPNDAINFES NGNFIAPEYAYKIVKKGDSAIVKSEVEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSN KLVLATGLRNSPLRERRRKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKEST QKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENE RTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEAR LKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICI-GYIPEAPRDGQ AYVRKDGEWVLLSTFL-RSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 17 [HA1-Fd-hFc]:  ICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPM CDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGV SSACPYQGTPSFFRNVVWLIKKNNTYPTIKRSYNNTNQEDLLILWGIHHSNDAAEQTKLYQNPT TYISVGTSTLNQRLVPKIATRSKVNGQNGRMDFFWTILKPNDAINFESNGNFIAPEYAYKIVKKG DSAIVKSEVEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNKLVLATGLRNSPL-GYI PEAPRDGQAYVRKDGEWVLLSTFL-RSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK SEQ ID NO. 18 [HA2-Fd-hFc]:  GLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQ FEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQ LRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSI YSTVASSLALAIMVAGLSLWMCSNGSLQCRICI-GYIPEAPRDGQAYVRKDGEWVLLSTFL-RS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 19  LSRINHFEKIQIIPKSSWSDHEASSGVSSACPYQGTPSFFRNVVWLIKKNNTYPTIKRSYNNTNQ EDLLILWGIHHSNDAAEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQNGRMDFFWTILK PNDAINFESNGNFIAPEYAYKIVKK-GYIPEAPRDGQAYVRKDGEWVLLSTFL-RSDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 20 [mouse loG Fc (mFc)]:  RSPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFV NNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGS VRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYF MYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO. 21  MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPL ILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKI QIIPKSSWSDHEASSGVSSACPYQGTPSFFRNVVWLIKKNNTYPTIKRSYNNTNQEDLLILWGIH HSNDAAEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQNGRMDFFWTILKPNDAINFES NGNFIAPEYAYKIVKKGDSAIVKSEVEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSN KLVLATGLRNSPLRERRRKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKEST QKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENE RTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEAR LKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICI-GYIPEAPRDGQ AYVRKDGEWVLLSTFL-RSPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCV VVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVN NKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTE LNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO. 22  ICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPM CDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGV SSACPYQGTPSFFRNVVWLIKKNNTYPTIKRSYNNTNQEDLLILWGIHHSNDAAEQTKLYQNPT TYISVGTSTLNQRLVPKIATRSKVNGQNGRMDFFWTILKPNDAINFESNGNFIAPEYAYKIVKKG DSAIVKSEVEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNKLVLATGLRNSPL-GYI PEAPRDGQAYVRKDGEWVLLSTFL-RSPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMI SLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSG KEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVE WTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFS RTPGK SEQ ID NO. 23  GLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQ FEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQ LRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSI YSTVASSLALAIMVAGLSLWMCSNGSLQCRICI-GYIPEAPRDGQAYVRKDGEWVLLSTFL-RS PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVN NVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSV RAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYF MYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO. 24  LSRINHFEKIQIIPKSSWSDHEASSGVSSACPYQGTPSFFRNVVWLIKKNNTYPTIKRSYNNTNQ EDLLILWGIHHSNDAAEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQNGRMDFFWTILK PNDAINFESNGNFIAPEYAYKIVKK-GYIPEAPRDGQAYVRKDGEWVLLSTFL-RSPRGPTIKPC PPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQT QTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLP PPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKK NWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO. 25 [rabbit IqG Fc (rFc)]:  RSSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRT ARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYT MGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVP TSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK SEQ ID NO. 26  MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPL ILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKI QIIPKSSWSDHEASSGVSSACPYQGTPSFFRNVVWLIKKNNTYPTIKRSYNNTNQEDLLILWGIH HSNDAAEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQNGRMDFFWTILKPNDAINFES NGNFIAPEYAYKIVKKGDSAIVKSEVEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSN KLVLATGLRNSPLRERRRKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKEST QKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENE RTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEAR LKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICI-GYIPEAPRDGQ AYVRKDGEWVLLSTFL-RSSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQD DPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIE KTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAV LDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK SEQ ID NO. 27  ICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPM CDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGV SSACPYQGTPSFFRNVVWLIKKNNTYPTIKRSYNNTNQEDLLILWGIHHSNDAAEQTKLYQNPT TYISVGTSTLNQRLVPKIATRSKVNGQNGRMDFFWTILKPNDAINFESNGNFIAPEYAYKIVKKG DSAIVKSEVEYGNCNTKCQTPIGAINSSMPFHNIHPLTIGECPKYVKSNKLVLATGLRNSPL-GYI PEAPRDGQAYVRKDGEWVLLSTFL-RSSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTC VVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVH NKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAE DNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK SEQ ID NO. 28  GLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQ FEAVGREFNNLERRIENLNKKMEDGFLDWVTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQ LRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSI YSTVASSLALAIMVAGLSLWMCSNGSLQCRICI-GYIPEAPRDGQAYVRKDGEWVLLSTFL-RS SKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTAR PPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMG PPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSE WQRGDVFTCSVMHEALHNHYTQKSISRSPGK SEQ ID NO. 29  LSRINHFEKIQIIPKSSWSDHEASSGVSSACPYQGTPSFFRNVVWLIKKNNTYPTIKRSYNNTNQ EDLLILWGIHHSNDAAEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQNGRMDFFWTILK PNDAINFESNGNFIAPEYAYKIVKK-GYIPEAPRDGQAYVRKDGEWVLLSTFL-RSSKPTCPPPE LLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFN STIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRS VSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTC SVMHEALHNHYTQKSISRSPGK SEQ ID NO. 30 [A/Anhui/1/2005(H5N1) HA1 +3-259]:  ICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPM CDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGV SSACPYQGTPSFFRNVVWLIKKNNTYPTIKRSYNNTNQEDLLILWGIHHSNDAAEQTKLYQNPT TYISVGTSTLNQRLVPKIATRSKVNGQNGRMDFFWTILKPNDAINFESNGNFIAPEYAYKIVKK SEQ ID NO. 31 [HA1 +3-259-Fd]:  ICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPM CDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGV SSACPYQGTPSFFRNVVWLIKKNNTYPTIKRSYNNTNQEDLLILWGIHHSNDAAEQTKLYQNPT TYISVGTSTLNQRLVPKIATRSKVNGQNGRMDFFWTILKPNDAINFESNGNFIAPEYAYKIVKK- GYIPEAPRDGQAYVRKDGEWVLLSTFL SEQ ID NO. 32 [HA1 +3-259-hFc]:  ICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPM CDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGV SSACPYQGTPSFFRNVVWLIKKNNTYPTIKRSYNNTNQEDLLILWGIHHSNDAAEQTKLYQNPT TYISVGTSTLNQRLVPKIATRSKVNGQNGRMDFFWTILKPNDAINFESNGNFIAPEYAYKIVKK- RSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 33 [HA1 +3-259-Fd-hFc]:  ICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPM CDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGV SSACPYQGTPSFFRNVVWLIKKNNTYPTIKRSYNNTNQEDLLILWGIHHSNDAAEQTKLYQNPT TYISVGTSTLNQRLVPKIATRSKVNGQNGRMDFFWTILKPNDAINFESNGNFIAPEYAYKIVKK- GYIPEAPRDGQAYVRKDGEWVLLSTFL-RSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK SEQ ID NO. 34 [HA1 +3-259-Fd-mFc]:  ICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPM CDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGV SSACPYQGTPSFFRNVVWLIKKNNTYPTIKRSYNNTNQEDLLILWGIHHSNDAAEQTKLYQNPT TYISVGTSTLNQRLVPKIATRSKVNGQNGRMDFFWTILKPNDAINFESNGNFIAPEYAYKIVKK- GYIPEAPRDGQAYVRKDGEWVLLSTFL-RSPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDV LMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDW MSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDI YVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTK SFSRTPGK SEQ ID NO. 35 [HA1 +3-259-Fd-rFc]:  ICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPM CDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGV SSACPYQGTPSFFRNVVWLIKKNNTYPTIKRSYNNTNQEDLLILWGIHHSNDAAEQTKLYQNPT TYISVGTSTLNQRLVPKIATRSKVNGQNGRMDFFWTILKPNDAINFESNGNFIAPEYAYKIVKK- GYIPEAPRDGQAYVRKDGEWVLLSTFL-RSSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPE VTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKC KVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNG KAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

In one embodiment, the stabilization sequence comprises a sequence that stabilizes the HA sequence in the trimer or oligomer configuration. As used herein, the terms stabilization sequence, trimeric motif and trimerization sequence are interchangeable and equivalent. Suitable stabilization sequences include, but are not limited to, foldon a 27 amino acid region of the C-terminal domain of T4 fibritin (GYIPEAPRDGQAYVRKDGEWVLLSTFL, SEQ ID NO:6 or GSGYIPEAPRDGQAYVRKDGEWVLLSTFL, SEQ ID NO:36), GCN4 (MKQIEDKIEEILSKIYHIENEIARIKKLIGEV; SEQ ID NO. 37), IQ (RMKQIEDKIEEIES KQKKIENEIARIKK; SEQ ID NO. 38) or IZ (IKKEIEAIKKEQEAIKKKIEAIEK; SEQ ID NO. 39). Other suitable stabilization methods include, but are not limited to, 2,2-bipyridine-5-carboxylic acid (BPY), disulfide bonds and facile ligation.

In another embodiment, the immunopotentiator comprises a sequence to enhance the immunogenicity of the immunogenic composition. Suitable immunopotentiators include, but are not limited to, the Fc fragment of human IgG, C3d (a complement fragment that promotes antibody formation binding to antigens enhancing their uptake by dendritic cells and B cells), ASP-1 (Onchocerca volvulus homologue of the activation associated secreted gene family) (see US 20060039921, which is incorporated by reference herein for all it discloses regarding ASP-1 adjuvants), cholera toxin, muramyl peptides and cytokines.

In one embodiment, the claimed fusion proteins can be constructed using overlapping primers. In another embodiment, the DNA sequence (GGCTATATTCCG GAAGCGCCGCGTGATGGCCAGGCGTATGTGCGTAAAGATGGCGAATGGGTGCTG CTGTCTACCTTTCTG; SEQ ID NO:40) encoding Fd is synthesized first. Separate PCR products of HA1 and HA-3-259 and Fd are generated and the HA1-Fd and HA-3-259-Fd fusion fragment is amplified by one-round PCR using an HA1 or HA-3-259 Forward primer and Fd Reverse primer with HA1 and Fd DNA (PCR products) as templates. The amplified HA1-Fd and HA-3-259-Fd PCR products are then inserted into the hFc vector, to produce HA1-Fd-hFc and HA-3-259-Fd-hFc recombinant plasmids encoding HA1-Fd-hFc and HA-3-259-Fd-hFc fusion proteins, respectively.

In one embodiment, pFUSE-hIgG1-Fc (human Fc, hFc), pFUSE-mlgG2a-Fc2 (murine Fc, mFc), or pFUSE-rIgG2-Fc2 (rabbit Fc, rFc) vectors are used for construction of the disclosed fusion proteins. In another embodiment, the fusion proteins can be expressed from other mammalian cell expression vectors, including, but not limited to, pcDNA3.1, pcDNA6-His, PEE13.1, PEE1.41, pCMV-NEO-BAM, pSV2, and pCMV1, 2, 3, 4, 5, 6. In another embodiment, the fusion proteins can be expressed from insect cell expression vectors including, but not limited to, pAcGP67, pFastBac Dual, and pMT/V5-His-TOPO. In yet another embodiment, the fusion proteins can be expressed from E. coli expression vectors including, but not limited to, pET, pET-SUMO, and pGEX vectors with GST.

The following expression systems are suitable for use in expressing the disclosed fusion proteins: mammalian cell expression systems such as, but not limited to, the pcDNA and GS Gene expression systems; insect cell expression systems such as, but not limited to, Bac-to-Bac, baculovirus and DES expression systems; and E. coli expression systems including, but not limited to, pET, pSUMO and GST expression systems.

Advantages of proteins expressed in mammalian cell expression systems include the follows. The mammalian cell expression system is a relatively mature eukaryotic system for expression of recombinant proteins. It is more likely to achieve correctly folded soluble proteins with proper glycosylation, making the expressed protein maintain its native conformation and keep sufficient bioactivity. This system can either transiently or stably express recombinant antigens, and promote signal synthesis. Recombinant proteins expressed in this way may keep good antigenicity and immunogenicity. However, both insect and bacterial expression systems provide inexpensive and efficient expression of proteins which may be appropriate under certain conditions.

The purification systems are dependent on whether a tag is linked or fused with the HA proteins. When the fusion proteins are fused with IgG Fc vectors, Protein A or Protein G affinity chromatography is used for the purification. If the fusion proteins are fused with GST proteins, the GST columns will be used for the purification. If the fusion proteins link with 6×His tag at the N- or C-terminal, the expressed proteins are be purified using His tag columns. If no tag is linked with recombinant proteins, the expressed proteins could be purified using Fast protein liquid chromatography (FPLC), High performance liquid chromatography (HPLC) or other chromatography.

In certain embodiments, the immunogenic compositions further comprise or are administered with an adjuvant. Adjuvants suitable for use in animals include, but are not limited to, Freund's complete or incomplete adjuvants, Sigma Adjuvant System (SAS), and Ribi adjuvants. Adjuvants suitable for use in humans include, but are not limited to, MF59 (an oil-in-water emulsion adjuvant), Montanide ISA 51 or 720 (a mineral oil-based or metabolizable oil-based adjuvant), aluminum hydroxide, -phosphate or -oxide, HAVLOGEN® (an acrylic acid polymer-based adjuvant, Intervet Inc., Millsboro, Del.), polyacrylic acids, oil-in-water or water-in-oil emulsion based on, for example a mineral oil, such as BAYOL™ or MARCOL™ (Esso Imperial Oil Limited, Canada), or a vegetable oil such as vitamin E acetate, saponins, and Onchocerca volvulus activation-associated protein-1 (ASP-1) (see US 20060039921, which is incorporated by reference herein for all it discloses regarding ASP-1 adjuvants). However, components with adjuvant activity are widely known and, generally, any adjuvant may be utilized that does not adversely interfere with the efficacy or safety of the vaccine and/or immunogenic composition.

Vaccine and immunogenic compositions according to the various embodiments disclosed herein can be prepared and/or marketed in the form of a liquid, frozen suspension or in a lyophilized form. Typically, vaccines and/or immunogenic compositions prepared according to the present disclosure contain a pharmaceutically acceptable carrier or diluent customarily used for such compositions. Carriers include, but are not limited to, stabilizers, preservatives and buffers. Suitable stabilizers are, for example SPGA, Tween compositions (such as are available from A.G. Scientific, Inc., San Diego, Calif.), carbohydrates (such as sorbitol, mannitol, starch, sucrose, dextran, glutamate or glucose), proteins (such as dried milk serum, albumin or casein) or degradation products thereof. Non-limiting examples of suitable buffers include alkali metal phosphates. Suitable preservatives are thimerosal, merthiolate and gentamicin. Diluents include water, aqueous buffer (such as buffered saline), alcohols and polyols (such as glycerol).

Also disclosed herein are methods for inducing an immune response to an influenza virus using the disclosed fusion proteins. Generally, the vaccine or immunogenic composition may be administered subcutaneously, intradermally, submucosally, or intramuscularly in an effective amount to prevent infection from the influenza virus of interest and/or treat an infection from the influenza virus. An effective amount is defined as an amount of immunizing fusion protein that will induce immunity in the vaccinated animals, against challenge by a virulent virus. Immunity is defined herein as the induction of a significant higher level of protection in a population of the animal after vaccination compared to an unvaccinated group.

Further, in various formulations of the vaccines and/or immunogenic compositions, suitable excipients, stabilizers and the like may be added.

EXAMPLES Example 1 Construction and Expression of Recombinant HA Proteins of H5N1 Virus

Construction of recombinant plasmids encoding HA1-hFc, HA1-Fd-hFc and HA-3-259-Fd-hFc. The genes encoding the fragment containing 320 amino acid (aa) (+3-322) of the HA1 fragment (SEQ ID NO. 2) of the H5N1 HA protein were amplified by PCR with a plasmid containing codon-optimized full-length HA (SEQ ID NO. 1) of influenza A H5N1 virus [A/Anhui/1/2005(H5N1), GenBank accession number ABD28180.1)] as the template, and fused in frame into the pFUSE-hIgG1-Fc2 (human IgG Fc, hFc) expression vector (InvivoGen, San Diego, Calif.). The constructed recombinant plasmid was thus named pHA1-hFc. The Fd sequence were added at the 3′ end by PCR using overlapping primers covering Fd, followed by insertion into the above hFc expression vector, which was named pHA1-Fd-hFc. The plasmid coding HA-3-259-Fd-hFc (SEQ ID NO:33) was constructed by insertion of genes encoding the fragment containing residues +3-259 of the above HA1 fragment (SEQ ID NO. 2) of H5N1 HA protein plus the above Fd sequence into the above hFc expression vector. The constructed recombinant plasmids were confirmed by sequencing analysis. The aa sequences of the H5N1 HA proteins, Fd and hFc fragments are listed in the Table 1.

The HA protein of A/Anhui/1/2005(H5N1) virus contains fragments of the signal peptide (SP), HA1 (+1-329 aa) and HA2 (+330-551 aa) spanned by a specific sequence of protease cleavage site RERRRKR (SEQ ID NO:41) between HA1 and HA2. In the construction of the recombinant HA1-hFc plasmid, the original signal peptide of the HA protein of H5N1 virus was replaced by the IL2ss signal sequence (SEQ ID NO:5), which was followed by HA1 fragment of H5N1 (+3-322 aa) fused into the above hFc vector. The Fd sequence was inserted between HA1 and hFc, becoming HA1-Fd-hFc (SEQ ID NO:17). HA-3-259-Fd-hFc contains +3-259 aa of the above HA1 fragment of H5N1 plus the Fd sequence, fusing into the above hFc vector.

Expression, purification and characterization of recombinant HA1-hFc, HA1-Fd-hFc and HA-3-259-Fd-hFc proteins. The recombinant HA1-hFc, HA1-Fd-hFc and HA-3-259-Fd-hFc proteins were expressed as previously described (Du L et al. Biochem Biophys Res Commun 384, 486-490, 2009). In brief, recombinant plasmids encoding HA1-hFc, HA1-Fd-hFc and HA-3-259-Fd-hFc proteins were transfected into mammalian 293T cells (ATCC, Manassas, Va.) seeded 24 hr prior to transfection using the calcium phosphate method. Culture medium was replaced by fresh OPTI-MEM I Reduced-Serum Medium (Invitrogen, Carlsbad, Calif.) 10 hr later, and supernatant was collected 72 hr post-translation. The recombinant HA1-hFc, HA1-Fd-hFc and HA-3-259-Fd-hFc proteins in the supernatant were purified by Protein A affinity chromatography (GE Healthcare, Piscataway, N.J.). Conformational and characteristic analyses of HA1-hFc, HA1-Fd-hFc and HA-3-259-Fd-hFc proteins were performed by FPLC using AKTApurifier Core Systems and Unicon 5.11 software according to manufacturer's protocols (GE Healthcare Life Sciences).

Detection of protein expression by SDS-PAGE and Western blot. The purified proteins were analyzed by SDS-PAGE and Western blot as our previously described protocols (Du L et al. Virology. 393, 144-150, 2009) using HA-specific monoclonal antibodies (mAbs). In brief, 10 μg of purified proteins was separated by 10-20% Tricine SDS-PAGE gels (Invitrogen) and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, Calif.). After blocking overnight at 4° C., the blots were incubated with a HA-specific mAb at 1:1,000 dilution for 1 hr at room temperature. After three washes with PBS containing 0.1% Tween-20 (PBST), the blots were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:5,000, Zymed, Carlsbad, Calif.) for 1 hr at room temperature. Signals were visualized with ECL Western blot substrate reagents and Amersham Hyperfilm (GE Healthcare).

As shown in FIG. 2, the recombinant HA1-hFc and HA1-Fd-hFc proteins could be expressed at a very high level in the secreted form in the culture supernatant of transfected 293T cells and were purified with high purity (FIG. 2a ). They were recognized by the conformation-specific mAb against HA protein of H5N1 viruses as detected by Western blot (FIG. 2b ), indicating that these proteins are specific to HA of influenza A H5N1 virus, suggesting that these fusion proteins maintained proper conformation and antigenicity of H5N1 viral protein HA. Further characterization of these proteins by FPLC analysis demonstrated that the majority of HA1 protein fused with Fc (HA1-hFc) mainly formed a trimer with a molecule size of ˜440 kDa, while HA1 protein fused with Fd and Fc (HA1-Fd-hFc) mainly constituted an oligomeric structure with higher molecular weight at ˜669 kDa (FIG. 3).

Example 2 Detection of Humoral Immune Responses Induced by Recombinant HA Fusion Proteins

Groups of female BALB/c mice, age 4-6 weeks, were vaccinated subcutaneously (s.c.) with 20 μg/mouse of purified HA fusion proteins re-suspended in PBS in the presence of Sigma Adjuvant System (SAS, Sigma) and boosted three times with 10 μg/mouse of immunogen containing SAS at approximate 3-week intervals. PBS plus SAS was used as the negative control. Serum samples were collected before immunization and 10 days post-each vaccination to detect the generation of HA- and/or H5N1 virus-specific IgG antibodies and subtypes using ELISA. The immunization scheme is described in Table 2 and FIG. 4.

Enzyme-linked immunosorbent assays (ELISA) was used to evaluate IgG antibody responses and subtypes induced by HA proteins as previously described (Du L et al. Vaccine 25, 2832-2838, 2007; Du L et al. Virology 393:144-150, 2009). Briefly, 96-well microtiter plates were pre-coated respectively with the recombinant HA1-hFc, HA1-Fd-hFc and HA-3-259-Fd-hFc proteins, HA1 protein without Fd and hFc, and/or inactivated H5N1 virus A/VietNam/1194/2004 (VN/1194) overnight at 4° C. and blocked with 2% non-fat milk at 37° C. for 2 hr. Serially diluted mouse sera were added to the plates and incubated at 37° C. for 1 hr, followed by four washes with PBST. Bound antibodies were then reacted with HRP-conjugated goat anti-mouse IgG (Zymed), IgG1 (Invitrogen), and/or IgG2a (Bethyl Laboratories, Montgomery, Tex.) for 1 hr at 37° C. After four washes, the substrate 3,3′,5,5′-tetramethylbenzidine (TMB) (Zymed) was added to the plates, and the reaction was stopped by adding 1 N H₂SO₄. The absorbance at 450 nm (A450) was measured by an ELISA plate reader (Tecan, San Jose, Calif.).

Antibody levels induced by HA1-hFc, HA1-Fd-hFc and HA-3-259-Fd-hFc proteins were detected by ELISA against HA fusion proteins. As shown in FIG. 5, both HA1-hFc and HA1-Fd-hFc proteins induced IgG antibody responses specific to the purified HA1-hFc and HA1-Fd-hFc proteins, quickly reaching a high level after the first boost vaccination, then slightly increasing antibody binding after each boost (sera were tested at a dilution of 1:3,000), while only background levels of antibody responses was detected in sera collected from prior immunization (pre-immune) and those from PBS control. An average end-point antibody titer of 1:2.1×10⁵ was detected in mouse sera collected at 10 days post last boost (FIG. 6). The mean titer of the IgG antibodies in the sera, collected after the last boost, of the mice immunized with HA1-hFc, HA1-Fd-hFc and HA-3-259-Fd-hFc reached 1:3.9×10⁷±2.2×10⁷, 1:1.5×10⁸±8.6×10⁷, and 1:2.1×10⁵, respectively (FIG. 7). The data in FIGS. 5-7 are expressed as Mean±Standard Deviation (SD).

Antibody levels were further detected by ELISA against a HA1 protein without Fd and hFc to eliminate the antibody response potentially induced by the fusion tag Fd and/or hFc and, in addition against an inactivated heterologous H5N1 virus (VN/1194). As illustrated in FIG. 8, sera of mice vaccinated with these HA fusion proteins, particularly HA1-Fd-hFc, reacted strongly with HA1 proteins without Fd and/or Fc, reaching an end-point titer of 1:1.3×10⁷, which suggests the high specificity of the antibody responses to the HA1 protein. It was further shown that the induced IgG antibodies could also react with an inactivated H5N1 virus (VN/1194), reaching a similar end-point titer of 1:1.3×10⁷ (FIG. 9). However, no IgG antibody response was detectable in the sera of control mice injected with PBS (FIGS. 8 and 9).

The evaluation of IgG subtypes induced by HA1-hFc and HA1-Fd-hFc proteins showed that IgG1 and IgG2a were detectable in the mouse sera collected at 10 days post last vaccination. Both HA1-hFc and HA1-Fd-hFc proteins elicited similar levels of IgG1 (Th2-associated, FIG. 10) and IgG2a (Th1-associated, FIG. 11) antibody responses specific to the HA1 proteins, reaching an end-point titer of 1:2.1×10⁸. FIG. 12 further demonstrated that the IgG2a antibody titer was significantly higher than IgG1 (P<0.05), suggesting that both HA1-hFc and HA1-Fd-hFc fusion proteins have a tendency to stimulate Th1-associated antibody responses. However, no IgG1 or IgG2a antibody responses were found in sera of PBS control mice (FIGS. 10-12).

The above data suggest that expressed HA1-hFc, HA1-Fd-hFc and HA-3-259-Fd-hFc proteins are able to elicit high titers of antibody responses specific to the HA1 proteins of homogeneous and/or heterogeneous H5N1 viruses, implying their strong immunogenicity in stimulating highly potent humoral immune responses in the vaccinated mice. The data in FIGS. 8-12 are expressed as Mean±SD.

TABLE 2 Immunization scheme of recombinant HA fusion proteins for detection of antibody responses* 1^(st) immunization Boost 1 Boost 2 Boost 3 Group (Day 0) (Day 21) (Day 42) (Day 63) 12A (N = 3) 20 μg protein in 10 μg protein in 10 μg protein in 10 μg protein in 4 doses PBS + PBS + PBS + PBS + HA1-hFc 100 μL SAS 100 μL SAS 100 μL SAS 100 μL SAS 12B (N = 5) 20 μg protein in 10 μg protein in 10 μg protein in 10 μg protein in 4 doses PBS + PBS + PBS + PBS + HA1-Fd-hFc 100 μL SAS 100 μL SAS 100 μL SAS 100 μL SAS 12C (N = 5) 20 μg protein in 10 μg protein in 10 μg protein in 10 μg protein in 4 doses PBS + PBS + PBS + PBS + HA336-89Fd-Fc 100 μL SAS 100 μL SAS 100 μL SAS 100 μL SAS 12D (N = 3) 20 μg protein in 10 μg protein in 10 μg protein in 10 μg protein in 4 doses PBS + PBS + PBS + PBS + HA2-89Fc 100 μL SAS 100 μL SAS 100 μL SAS 100 μL SAS 12E (N = 4) 20 μg protein in 10 μg protein in 10 μg protein in 10 μg protein in 4 doses PBS + PBS + PBS + PBS + HA2-89Fd-Fc 100 μL SAS 100 μL SAS 100 μL SAS 100 μL SAS 19C (N = 5) 20 μg protein in 10 μg protein in 10 μg protein in 10 μg protein in 4 doses PBS + PBS + PBS + PBS + HA-3-259-Fd-hFc 100 μL SAS 100 μL SAS 100 μL SAS 100 μL SAS *All immunizations were in 200 μL total volume; SAS = Sigma Adjuvant System (Sigma). Blood samples were collected on day 0 (pre-immune) and 10 days after each boost for detection of antibody responses.

Example 3 Detection of Neutralizing Antibody Activity Induced by Recombinant HA Fusion Proteins Using H5N1 Pseudovirus Neutralization Assay

The fusion proteins HA1-hFc and HA1-Fd-hFc were then evaluated for generation of neutralizing antibodies against highly pathogenic avian influenza (HPAI) H5N1 viruses based on a pseudotype neutralization assay. Sera were collected at pre-immunization and 10 days after each vaccination. Sera collected from Group12A (HA1-hFc), Group12B (HA1-Fd-hFc) and Group19C (HA-3-259-Fd-hFc) were tested for generation of neutralizing antibody activity against H5N1 pseudovirus expressing HAs of five isolates covering four clades, including the homologous strain A/Anhui/1/2005 (AH-HA, clade 2.3), and heterologous strains, such as A/Hong Kong/156/97 (HK-HA, clade 0), A/VietNam/1194/2004 (1194-HA, clade 1), A/Qinghai/59/05 (QH-HA, clade 2.2) and A/Xinjiang/1/2006 (XJ-HA, clade 2.2).

H5N1 Pseudovirus Production.

The generation of H5N1 pseudovirus was done as previously described (Du L et al. Virology. 393:144-150, 2009; Du L et al. Biochem Biophys Res Commun 384:486-490, 2009; Du L et al. Biochem Biophys Res Commun 397:580-585, 2010) with some modifications. In brief, 293T cells were co-transfected with a plasmid encoding the HA of influenza A virus H5N1 isolates HK, 1194, QH, XJ, and AH, and a plasmid encoding the Env-defective, luciferase-expressing HIV-1 genome (pNL4-3.1uc.RE) using the calcium phosphate method. The medium, Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), was changed 10 hr later and neuraminidase (NA) (Sigma) was added to the culture medium 26 and 50 hr post-transfection at concentrations of 0.5-5 μg/ml. Supernatants were harvested 72 hr post-transfection and used for single-cycle infection of 293T cells.

Neutralizing antibody activity detected by H5N1 pseudovirus. In the detection of neutralizing activity of vaccinated mouse sera, all serum samples were heat-inactivated at 56° C. for 30 min and diluted in serial dilutions. An equal volume of samples and H5N1 pseudovirus were added to each well and incubated for 1 hr at 37° C. Then 100 μL of this mixture was added to each well of a 96-well tissue culture plate plated with 293T cells 6-8 hr previously. Twenty-four hours later, 80 μL/well of fresh DMEM containing 10% FBS was added to the wells and luciferase activity was detected 72 hr later. Cells were lysed using cell lysis buffer (Promega, Madison, Wis.). After addition of luciferase substrate (Promega), relative luciferase activity was determined in Ultra 384 luminometer (Tecan). The 50% neutralizing antibody titer (NT₅₀)) was calculated using Calcusyn program (Chou, T. C. Pharmacol Rev 58:621-681, 2006).

The experimental results for the detection of neutralizing activity against H5N1 pseudovirus showed that these fusion proteins were able to induce highly potent specific IgG antibodies with neutralizing activity against H5N1 pseudovirus. Starting from 10 days post 1^(st) boost vaccination, neutralizing antibodies were detected in mouse sera vaccinated with both HA1-hFc (FIG. 13) and HA1-Fd-hFc (FIG. 14). No, or low (NT₅₀) 1:50), levels of neutralizing antibodies were detected in pre-vaccinated mouse sera (FIGS. 13 and 14). Serum levels of these neutralizing antibodies increased rapidly at each time post-boost vaccination, reaching the highest level at 10 days post last vaccination (FIG. 15). The data in FIGS. 13-15 are expressed as Mean±SD.

The above induced high titers of neutralizing antibodies could not only neutralize homologous strain of AH-HA (FIG. 15), but also neutralized heterologous HK-HA, 1194-HA, XJ-HA, and even QH-HA stains (FIGS. 13, 14, and 16), suggesting their potential ability in inducing cross-protection against divergent H5N1. In general, both proteins fused with Fc of hIgG1 (HA1-hFc and HA1-Fd-hFc) could induce potent neutralizing antibodies against H5N1 pseudovirus infection in 293T cells (FIGS. 14-16, Table 3), suggesting that IgG Fc may play a key role in the formation of oligomer structures of HA proteins and the enhancement of the immunogenicity of the fusion proteins. In addition, the HA1-Fd-hFc fusion protein with Fd sequences could induce higher titers of pseudovirus neutralizing antibodies than HA1-hFc protein without Fd, showing a significantly higher level of inhibition against infection by 1194-HA, QH-HA, XJ-HA and AH-HA H5N1 pseudoviruses (FIG. 16, P<0.05). The induction of high titers of neutralizing antibodies against infections of HK-HA, 1194-HA, and AH-HA H5N1 pseudoviruses were also detected in the sera of mice collected at 10 days post last vaccination of HA-3-259-Fd-hFc protein (FIG. 17), furthering confirming the importance of Fd sequences in the induction of highly potent neutralizing antibodies against divergent strains of H5N1 pseudoviruses. The Fd sequences may be helpful to form trimer structures of HA proteins, thus increasing neutralizing ability. The above results also suggest that in addition to the HA1 fragment containing residues +3-322, a shorter HA1 fragment of H5N1 covering residues +3-259 contains important neutralizing epitopes that induce highly potent neutralizing antibodies against multiple strains of H5N1 viruses. In contrast, the PBS control group only elicited a background level of neutralizing antibody titers against the tested H5N1 pseudoviruses (FIG. 16). The data in FIGS. 16 and 17 are expressed as Mean±SD.

TABLE 3 Pseudovirus neutralizing antibody titer (NT₅₀) in sera of mice vaccinated with HA fusion proteins* NT₅₀ of sera Influenza A virus of mice vaccinated with H5N1 strain HA1-hFc HA1-Fd-hFc HK-HA 3,323 ± 1,677 3,353 ± 1,476 1194-HA 443 ± 300 5,341 ± 1,475 XJ-HA 126 ± 119 6,963 ± 5,270 QH-HA 50 ± 3  350 ± 254 AH-HA 4,024 ± 996   38,177 ± 14,896 *Samples were from sera of mice collected at 10 days post last vaccination. The data are expressed as Mean ± SD of three to five mouse sera per group.

Example 4 Detection of Neutralizing Antibody Activity and Inhibition Induced by Recombinant HA Fusion Proteins Using H5N1 Live Virus Neutralization Assay and Hemagglutination Inhibition Assay

The fusion proteins HA1-hFc and HA1-Fd-hFc were further evaluated for generation of neutralizing antibodies against highly pathogenic H5N1 viruses based on a live virus neutralization assay and hemagluttinin inhibition. Sera collected from Group12A (HA1-hFc) and Group12B (HA1-Fd-hFc) at 10 days post last vaccination were applied for detection of neutralizing antibody activity against H5N1 live viruses.

H5N1 virus neutralization assay. Titers of neutralizing antibodies of vaccinated mice were further detected by live neutralization assay. In brief, serial two-fold diluted mouse sera were mixed with 20 plaque forming units (PFU) of clade 0: A/Hong Kong/156/97 (HK/156), clade 1: A/VietNam/1194/2004 (VN/1194) and clade 2.3.4: A/Shenzhen/406H/06 (SZ/406H) (H5N1) and incubated at 37° C. for 1 hr before adding to Madin-Darby canine kidney (MDCK) cells. Medium was replaced with fresh DMEM 1 hr later, and cell culture was continued for 72 hr at 37° C. The viral cytopathic effect (CPE) was observed daily and recorded on day 3 post-infection. The neutralizing antibody titer was determined based on the highest dilution of each serum, which completely suppressed CPE induced by the virus in >50% of the wells.

The hemagglutination inhibition (HI) assay. This assay was carried out as follows. In brief, serial dilutions of mouse sera at 10 days post last vaccination were incubated with equal volumes of HK/156, VN/1194 and/or SZ/406H H5N1 virus, for 1 hr at room temperature, followed by addition of equal volumes of 0.5% chicken red blood cells for 30 min at room temperature. The HI antibody titers were expressed as the highest serum dilution that completely inhibited hemagglutinating activity.

The experimental results for the detection of neutralizing activity against H5N1 live virus showed that the induced antibodies could neutralize infections of at least three heterologous strains covering three clades of H5N1 live viruses, such as HK/156 (clade 0), VN/1194 (clade 1) and SZ/406H (clade 2.3.4) (FIG. 18). In addition, the antibodies were able to inhibit the hemagglutination of these three H5N1 live viruses, with the average HI titer 1:1.0×10³ (FIG. 19). Notably, the neutralizing antibodies induced by HA1-Fd-hFc were generally greater than those induced by HA1-hFc. In contrast, the PBS control group only elicited a background level of neutralizing and HI antibody titers in the tested H5N1 live viruses (FIGS. 18 and 19). The data in FIGS. 18 and 19 are expressed as Mean±SD.

Example 5 H5N1 Virus Challenge and Cross-Protection Evaluation Induced by Recombinant HA Fusion Proteins Against Divergent Strains of H5N1 Virus

The fusion proteins HA1-hFc and HA1-Fd-hFc were then evaluated for inducing cross-protective immunity against highly pathogenic H5N1 viruses by observation of the survival rate of animals after H5N1 virus challenge, and detection of the viral load and histopathological changes in lung tissues collected from the mice at day 5 post-virus challenge.

H5N1 live virus challenge and sample collection. BALB/c female mice, 6-8 weeks old, were kept in biosafety level-3 (BSL-3) housing and given access to standard pellet feed and water ad libitum. All experimental protocols followed the standard operating procedures of the approved BSL-3 animal facilities and were approved by the Animal Ethics Committee. Mice (45 mice/group) were subcutaneously (s.c.) primed-vaccinated with 20 μg/mouse of purified HA1-hFc or HA1-Fd-hFc resuspended in PBS in the presence of SAS and boosted twice with 10 μg/mouse of immunogen containing SAS at 3-week intervals. Control mice were s.c. injected with the same volume of PBS-SAS. Mice were challenged intranasally (i.n.) with 10 LD₅₀ (50% Lethal Dose) of one of three clades of H5N1 virus, i.e., clade 0: HK/156, clade 1: VN/1194, and clade 2.3.4: SZ/406H, respectively (15 mice/group), at 10-12 days after the last vaccination. Infected mice were observed daily for 21 days or until the death of the mice for the survival rate detection. Five mice/group were sacrificed on day 5 post-challenge, and lung samples were collected for virological and histopathological detection. The immunization and virus challenge scheme is described in Table 4 and FIG. 4.

TABLE 4 Immunization scheme of recombinant HA fusion proteins for detection of cross-protective immunity Lung tissues Observe Vaccine Virus challenge 5 days post- two weeks Group Dosage 10-12 days post last vaccine challenge post-challenge 1A (N = 45) Vaccine A/Hong A/Vietnam/ A/Shenzhen/ 5 mice/group 10 mice/group 3 doses as Table 2 Kong/156/97 1194/04 406H/06 for viral for survival HA1-hFc 3 doses (15mice) (15mice) (15mice) load and rate 1B (N = 45) Vaccine A/Hong A/Vietnam/ A/Shenzhen/ histopathology 10 mice/group 3 doses as Table 2 Kong/156/97 1194/04 406H/06 analysis for survival HA1-Fd-hFc 3 doses (15mice) (15mice) (15mice) rate 1C (N = 45) Vaccine A/Hong A/Vietnam/ A/Shenzhen/ 10 mice/group 3 doses as Table 2 Kong/156/97 1194/04 406H/06 for survival PBS 3 doses (15mice) (15mice) (15mice) rate

Virological tests. Viral RNA in lung tissues was quantified by Q-RT-PCR as previously described (Zheng B J et al. Proc Natl Acad Sci USA 105:8091-8096. 2008). In brief, total RNA in lysed lung tissues was extracted by using RNeasy Mini kit (Qiagen, Valencia, Calif.) and reverse transcribed to cDNA by using applied SuperScript II Reverse Transcriptase (Invitrogen). Viral cDNA was synthesized by Superscript RT II (Invitrogen) using Uni12 primer (AGCAAAAGC; SEQ ID NO:42). Real-time PCR was performed on the LightCycler 480 system (Roche Applied Sciences) using SYBR Green I Master (Roche) with gene-specific primer pairs (for HK/156, forward primer: 5′-TGTCAAGAAAGGAGACTCAGC-3′ [SEQ ID NO:43], reverse primer: 5′-ACCATCTACCATTCCCTGC-3′ [SEQ ID NO:44]; for VN/1194, forward primer: 5′-ATACACCCTCTCACCATCGG-3′ [SEQ ID NO:45], reverse primer: 5′-ACCATCTACCATTCCCTGCC-3′ [SEQ ID NO:46]; for SZ/406H, forward primer: 5′-ATACACCCTCTCACCATCGG-3′ [SEQ ID NO:47], reverse primer: 5′-ACCATCTACCATTCCCTGC-3′ [SEQ ID NO:48]) targeting the H1 gene of different strains of H5N1 virus. The pcDNA3.1 plasmid, which contains the cloned H1 gene of the virus, was used as the standard.

Histopathological analysis. The lung tissues of challenged mice were immediately fixed in 10% buffered formalin and embedded in paraffin wax. Sections 4-6 μm in thickness were made and mounted on slides. Histopathological changes caused by H5N1 virus infection were examined by H&E staining and viewed under a light microscope as previously described (Du L et al. J Immunol 180:948-956. 2008; Zheng B J et al. Proc Natl Acad Sci USA 105:8091-8096. 2008).

The experimental results for the evaluation of cross-protection induced by recombinant HA fusion proteins against H5N1 live virus showed that all vaccinated mice survived challenge with HK/156 (clade 0, FIG. 20) and SZ/406H (clade 2.3.4, FIG. 22), suggesting that these two proteins may completely protect mice against challenges with different clades of H5N1 virus. All mice vaccinated with HA1-Fd-hFc also survived challenge with VN/1194 (clade 1), whereas about 10% of HA1-hFc-vaccinated mice did not survive challenge with this virus (FIG. 21). In contrast, no control mice injected with PBS survived the challenge with HK/156, VN/1194 and SZ/406H H5N1 viruses (FIGS. 20-22). These results demonstrated that vaccination with these fusion proteins, particularly HA1-Fd-hFc, could provide cross-clade protection against divergent strains of H5N1 virus infection.

The experimental results for the evaluation of cross-protection induced by recombinant HA fusion proteins against H5N1 live virus also demonstrated that viral RNA was undetectable in the HA1-hFc- and HA1-Fd-hFc-vaccinated mice challenged with VN/1194 virus, but a high level of viral RNA (8.6×10⁸ copies) was detected in the control mice injected with PBS. Lung tissues of mice vaccinated with HA1-hFc and HA1-Fd-hFc also exhibited significantly lower levels of viral RNA than the PBS control group after challenge with HK/156 and SZ/406H virus, respectively (P<0.05) (FIG. 23). The data in FIG. 23 are expressed as Mean±SD.

Examination of the H&E-stained lung tissues from virus-challenged mice revealed that all of the control mice injected with PBS developed a high degree of histopathological damage, including serious interstitial pneumonia and significant inflammation, which were characterized by predominant lymphocyte infiltration, epithelial cell degeneration, broadened interstitial spaces, pulmonary vascular dilatation and congestion, and focal hemorrhage and exudation. In contrast, mice receiving HA1-hFc and HA1-Fd-hFc vaccination neither developed significant pulmonary injury nor severe inflammation after challenge with all three H5N1 viruses covering different clades, showing lung structures similar to those of normal mice (FIG. 24). These results suggest that the immunity induced by the recombinant HA fusion proteins is able to highly suppress virus replication in vaccinated mouse lungs, indicating that vaccinations of HA1-hFc and HA1-Fd-hFc proteins reduced virus replication and limited lung damage in the mice infected by divergent strains of H5N1 virus.

The above results indicate the potential of the above tested candidate influenza vaccine in developing into a universal flu vaccine against divergent influenza viruses, suggesting its ability in prevention of future flu outbreak.

Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of” excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are inherently or expressly described and enabled herein.

Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above-cited references and printed publications are individually incorporated herein by reference in their entirety.

In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described. 

What is claimed is:
 1. A method of inducing a protective immune response against an influenza virus comprising: administering the immunogenic composition to a host in need thereof, wherein the immunogenic composition comprises a fusion polypeptide comprising: an immunogen sequence from the influenza virus; an immunopotentiator sequence comprising the Fc fragment of human IgG or a muramyl peptide; and a foldon stabilization sequence; wherein the immunogenic composition induces a protective immune response against challenge with infectious influenza virus in the host.
 2. The method according to claim 1, wherein the immunogenic composition further comprises an adjuvant.
 3. The method according to claim 1, wherein the immunogen is a hemagglutinin sequence of an influenza virus.
 4. The method according to claim 1, wherein the immunogen is a neuraminidase sequence of an influenza virus.
 5. The method according to claim 1, wherein the immunogen is a membrane protein sequence of the influenza virus.
 6. The method according to claim 1, wherein said polypeptide is produced in a mammalian expression system.
 7. The method according to claim 3, wherein the hemagglutinin sequence is a fragment of a hemagglutinin sequence selected from the group consisting of HA1, HA2 and RBD.
 8. The method according to claim 1, wherein the polypeptide is selected from the group consisting of HA1-hFc, HA-hFc, and HA1+3-259-hFc.
 9. The method according to claim 1, wherein the fusion polypeptide is selected from the group consisting of HA1-Fd-hFc, HA-Fd-hFc, HA2-Fd-hFc, HA-RBD-Fd-hFc, and HA1+3-259-Fd-hFc. 